LOCUS       sequence-197208-        8008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to LPAR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8008)
  AUTHORS   Baeumler TA, Ahmed AA, Fulga TA
  TITLE     Engineering Synthetic Signaling Pathways with Programmable 
            dCas9-Based Chimeric Receptors.
  JOURNAL   Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 
            10.1016/j.celrep.2017.08.044.
  PUBMED    28903044
REFERENCE   2  (bases 1 to 8008)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2017.08"; journalName: "Cell Rep"; date: 
            "2017-09-12- 12"; volume: "20"; issue: "11"; pages: "2639-2653"
COMMENT     Sequence Label: pLPAR1_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..8008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..574
                     /label=chicken beta-actin promoter
     intron          575..802
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2121..2147
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2163..2186,2187..2189,2190..3764)
                     /codon_start=1
                     /product="N-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the D10A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             3795..3851
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3858..3877)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4354..4374
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4491..4511)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4909..4920)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5051..5068)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5057..5264
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5273..5413
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5273..5402
                     /label=AAV2 ITR
     rep_origin      5488..5943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5575..5594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5785..5806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5960..5979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6079..6101
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6139..6157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6225..6329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6330..6398,6399..7190)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6548..6567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7361..7949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7850..7869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg ggggggggcg
      421 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
      481 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      541 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      601 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      661 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag
      721 gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct
      781 gcctgaaatc actttttttc aggttggacc ggtgcgccac catgaagacg atcatcgccc
      841 tgagctacat cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg
      901 atatggccgc tattagcacc agtatcccag ttatctctca gccccagttt acagcgatga
      961 atgagcctca gtgcttttac aacgaaagca tcgccttttt ctataacaga tccgggaagc
     1021 atctggccac cgaatggaat acggtgtcta aattggtcat gggccttggt attaccgttt
     1081 gcatctttat catgcttgca aacttgctgg tgatggtggc tatctacgtg aaccgcagat
     1141 tccattttcc aatttactac ttgatggcaa atcttgccgc agctgacttc ttcgcggggt
     1201 tggcatattt ctacttgatg tttaataccg ggcccaacac ccgcagactt actgtctcaa
     1261 cttggctgtt gcgccaggga ctcatcgaca cttccctgac tgcctctgtg gcaaacctgt
     1321 tggccatcgc aattgagaga cacataacgg tgttccgaat gcaacttcat acacggatgt
     1381 caaaccggag ggtggtggtg gtgatcgtgg tgatctggac catggctatc gttatgggcg
     1441 ccattcctag cgtgggttgg aattgcattt gcgacatcga gaactgttcc aatatggcac
     1501 ctctgtattc tgacagttat ctggttttct gggccatctt taatctcgtg acatttgtcg
     1561 tgatggtggt actgtatgct cacatcttcg gatacgtgag acaacgcaca atgcggatgt
     1621 ctcgacacag cagcggaccc agacggaatc gggacacaat gatgtccctc ctcaaaaccg
     1681 tcgtaatcgt gttgggcgca tttatcattt gttggacccc cgggctcgta ctgcttctgc
     1741 tcgacgtgtg ttgtccccag tgtgacgttc ttgcctatga gaagttcttt cttttgttgg
     1801 ccgaattcaa ctccgcaatg aatcctatta tttactctta tcgagataaa gagatgtccg
     1861 caacttttcg gcagatcctc tgctgccaga ggagcgagaa tcctacaggc cccactgaag
     1921 gttcagatcg gtcagcttca tccctcaacc acacaatcct ggccggtgta cactccaacg
     1981 atcattccgt ggtcatcgat accggtggac gcaccccacc cagcctgggt ccccaagatg
     2041 agtcctgcac caccgccagc tcctccctgg ccaaggacac ttcatcgacc ggtgagaacc
     2101 tgtacttcca gctaactagt tacccatacg acgtaccaga ttacgctgga ggttctggat
     2161 ccgacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga
     2221 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     2281 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     2341 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     2401 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     2461 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     2521 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     2581 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     2641 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     2701 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     2761 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     2821 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc
     2881 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     2941 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     3001 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     3061 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     3121 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     3181 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     3241 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     3301 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     3361 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     3421 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     3481 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     3541 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     3601 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     3661 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     3721 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaggtggc ggtggctcgg
     3781 gtaccggatc tggagccacc aatttttctc tgctgaagca ggctggtgat gttgaagaaa
     3841 accctggacc tgaattcatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     3901 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     3961 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     4021 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     4081 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     4141 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     4201 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     4261 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     4321 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     4381 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4441 gcaaggggta actaagtaag gatcgatcca agatatcgta ttcttaacta tgttgctcct
     4501 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4561 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4621 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4681 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     4741 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4801 ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc
     4861 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     4921 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     4981 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgccaat tcctagagct
     5041 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     5101 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     5161 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     5221 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc
     5281 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     5341 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     5401 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5461 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     5521 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     5581 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5641 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5701 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5761 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5821 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5881 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5941 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     6001 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     6061 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     6121 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     6181 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6241 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6301 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6361 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6421 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6481 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6541 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6601 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6661 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6721 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6781 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6841 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6901 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6961 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7021 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     7081 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7141 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7201 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7261 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7321 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7381 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7441 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7501 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7561 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7621 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7681 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7741 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7801 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7861 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7921 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7981 tggccttttg ctggcctttt gctcacat
//
